Effects of muscarine and adrenaline on neurones from Rana pipiens sympathetic ganglia.

Abstract

1. Neurones dissociated from Rana pipiens paravertebral sympathetic ganglia were studied by means of the whole-cell patch-clamp technique. Responses to agonists were best recorded when cyclic AMP was included in the patch pipette. 2. Two populations of cells were identified on the basis of size (input capacitance, Cin) and the presence or absence of a fast, transient outward current (A-current, IA). This current was usually present in the ''large'' cells (Cin = 40.5 .+-. 1.5 pF, n = 66) but absent from ''small'' cells (Cin = 21.0 .+-. 0.8 pF, n = 70). 3. Both cell types exhibited a slowly activating non-inactivating K+ current (M-current, IM) which was suppressed by luteinizng hormone-releasing hormone (LHRH, 10-100 .mu.M). Threshold for activation of IM was about -75 mV, half-maximal activation was at -50 mV and the M-conductance GM increased e-fold for a 7 mV change in membrane potential. The maximum value for IM studied in large cells by patch-clamp procedures was < 0.2 nA. More M-channels were available per unit membrane area in the small cells (.hivin.GM = 1495 .mu.S cm-2) than in the large cells (.hivin.GM = 1034 .mu.S cm-2). Time constants for IM deactivation at -70 mV were faster in the large cells (37.2 .+-. 4.6 ms, n = 16) than in the small cells (66.1 .+-. 5.9 ms, n = 9). 4. Muscarine (10 .mu.M) produced inward current in the large cells as a result of IM suppression. In 40% of the large cells, some of the M-channels were also sensitive to adrenaline (10-100 .mu.M). In a few large cells (< 10%) adrenaline produced outward current by increasing IM. 5. Muscarine failed to effect IM in the small cells and instead produced an inwardly rectifying K+ current which activated within 5 ms at -110 mV. The outward current produced in twenty out of thirty-seven small cells by adrenaline was occluded by that produced by muscarine, suggesting that both agonists affect the same K+ channels. 6. Inclusion of the protein kinase inhibitors, 1-(5-isoquinolinyl-sulphonyl)-2-methyl piperazine (H-7, 50 .mu.M) or gold sodium thiomalate (GST, 50 .mu.M) in the pipette solution failed to antagonize either muscarine-induced current. Both currents were prolonged when the "internal solution'' contained GTP-.gamma.-S (50 .mu.M). 7. Phorbol-12-myristate-13-acetate (PMA, 2-5 .mu.M) produced an inward current as a result of IM suppression in both small and large cells.